Protein in solution Radio waves 1 H 800 MHz 13 C 200 MHz 15 N 80 MHz Spectra Principles of NMR Protein Spectroscopy B o T Frequencies and Spectra

Principles of NMR Protein Spectroscopy How would the 1 H NMR spectrum appear? HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH AlaPhe ppm

Principles of NMR Protein Spectroscopy - NMR is very sensitive to environment HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH AlaPhe electronic, bonding small differences in for each nucleus in molecule   B 0 22 (Hz) s =  B 0 22 Actual General =  B 0 22 (Hz)  is shielding constant can or of nucleus -same for 13 C, 15 N Chemical Shift

Principles of NMR Protein Spectroscopy   B 0 22 (Hz) - resonance condition (field + local) s =  B 0 22 Chemical Shift B=B o (1-  ) =  B 0 22 recall  =  d +  p +  m +  r +  e +  s  d =   e 2 3m e r  e dr 0 8

Principles of NMR Protein Spectroscopy each nucleus has unique chemical shift HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH AlaPhe ppm similar for 13 C, 15 N 1 H spectrum

Principles of NMR Protein Spectroscopy Chemical Shift 4.80 ppm  = 10 6 sample - ref ppm ref ref = internal standard (0 ppm) T sample = MHz DSS  DSS = MHz  = 4.80 ppm Factors out B 0 -  is same at any B  DSS - dimethyl-silapentane-sulphonate 2877

Principles of NMR Protein Spectroscopy Chemical Shift DSS T, DSS = MHz T ref =  13 C * DSS  1 H =

Chemical Shifts of Proteins 1 H spectrum In general nuclei found in “ranges” of chemical shift ppm NH Aromatics HH H  CH ppm CC CC CH 3 13 C spectrum C  Principles of NMR Protein Spectroscopy

Protein Database BioMagResBank Reference Reference Information Chemical Shift Statistics Derived from the BMRB Database for: Restricted Set of Protein Chemical Shifts Full Set of Protein Chemical Shifts Restricted Set of DNA Chemical Shifts Full Set of DNA Chemical Shifts Restricted Set of RNA Chemical Shifts Full Set of RNA Chemical Shifts Principles of NMR Protein Spectroscopy

BioMagResBank actual chemical shifts for each amino acid are different dependence on placement in sequence and secondary structure Example HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH Ala Phe , 3.00 Principles of NMR Protein Spectroscopy

Spin-spin Coupling Principles of NMR Protein Spectroscopy     A AB =  B 0 22  B 0  22 +J J

Spin-spin Coupling Principles of NMR Protein Spectroscopy 1 H( 15 N) 1 H- 1 H 0-20 Hz 1 H- 13 C Hz 1 H- 15 N ~ 93 Hz 2nI +1 lines 13 C( 1 H) 93 Hz 143 Hz -CH 3 -NH

Spin-spin Coupling Principles of NMR Protein Spectroscopy 1 H- 1 H 0-20 Hz HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH 3J3J 3 J = 6.98cos 2  cos   =  - 60˚

Other Coupling Principles of NMR Protein Spectroscopy D = || (3cos 2  - 1) Dipolar Interaction 2 || = -  1  2 h 42r342r3 BoBo r 12 